This application is a 371 of PCT/JP99/06380 filed Nov. 16, 1999.
The present invention relates to a new strain of Bacillus subtilis. More specifically, the present invention relates to a Bacillus subtilis strain capable of efficiently producing vitamin K or a derivative thereof.
Heretofore, vitamin K is known to be an factor necessary for blood clotting or coagulation. Vitamin K is one of the fat-soluble vitamins and also called xe2x80x9cantihemorrhagic vitaminxe2x80x9d since the deficiency in vitamin K causes reduced coagulability of blood. It has been recently suggested that the reduced coagulability of blood may be caused by the deficiency of vitamin K since vitamin K is essential for the biosynthesis of several blood clotting factors including prothrombin. However, occurrence of vitamin K deficiency is rare, since only a minute amount of vitamin K of the order of xcexcg is required for preventing the reduction in coagulability of blood in a living body and vitamin K is usually supplied from intestinal bacteria in an adult human. Synthetic vitamin K1 or K2 has been used as a therapeutic for the treatment of vitamin K deficiency hemorrhagic disease, and a natural vitamin K1 concentrate has been used as a food for preventing the condition. Hence, vitamin K has been received less attention.
In recent years, however, it has become evident that vitamin K has both bone formation stimulative effect and bone resorption inhibitive effect and the administration of vitamin K can increase the bone density. On the other hand, osteoporosis is a condition characterized by brittleness of bone caused by aging, diseases or the like and associated with fracture or severe pain. Osteoporosis is being emerged as a major social problem from the viewpoint of medical care of the aged. It has been reported that patients with osteoporosis have a blood vitamin K level of about one-half that observed in normal individuals. In view these facts, synthetic vitamin K has been put in clinical tests as a therapeutic for osteoporosis. However, unlike the case of hemorrhagic disease, clinical tests demonstrate that, for the treatment and prevention of osteoporosis, administration of vitamin K in an amount as large as at least 45 mg per day is necessary for effective agglomeration of bone mass. For osteoporosis, prevention is more important than treatment after developing its symptoms. Therefore, it is desired to intake vitamin K through food on a day-to-day basis. However, it is yet unknown what amount of vitamin K should be taken by a normal individual per day for increasing the bone mass and for effective prevention of osteoporosis. Moreover, it seems to be difficult to take in such an amount of vitamin K as mentioned above through existing foods.
As mentioned above, it is desirable to take vitamin K through daily foods. In fact, vitamin K1 can be taken through green/yellow vegetables and seaweeds, and vitamin K2 can be taken through fermented foods such as xe2x80x9cnattoxe2x80x9d (a slippery fermented soybeans). However, if 45 mg of vitamin K (which is the amount reported to be effective for amelioration of osteoporosis) is taken through commercial foods, for example for a food containing 1 ppm of vitamin K, a large amount of 45 kg per day of the food should be taken, which is actually very difficult. A food which is most rich in vitamin K among the existing foods is natto, which contains a dozen ppm of vitamin K. However, even natto should be eaten in a large amount of several hundreds grams to several kilograms daily. It seems to be difficult to eat such a large amount of natto every day, from the viewpoint of liking. In addition, when vitamin K is orally taken in a single-shot manner, sufficient effect cannot be obtained due to the short half-life of vitamin K. In contrast, intake of a large amount of vitamin K at a time may cause a problem of adverse side-effects. Therefore, it is desirable to take vitamin K in a concentrated form. However, commercially available concentrates of natural vitamin K containing a powdered milk formula for the purpose of preventing hemorrhagic disease are expensive, while synthetic vitamin K produced for pharmaceutical purposes have not been yet recognized as food additives. For these reasons, addition of vitamin K concentrates to foods is impossible.
In the vitamin K group, only vitamin K1 and K2 naturally occur. Vitamin K1 is abundantly contained in foods, particularly in green vegetables, vegetable oils, seaweeds and so on. For example, seaweeds, layers and tea leaves contain several tens ppm, while soybean oil, spinach and broccoli contain several ppm. Alternatively, vitamin K, may be synthesized by the condensation of 2-methyl-1,4-naphthoquinone and phythyl acetate. As vitamin K2 compounds, there are known various homologues including menaquinone-1 to -14 (MK-1 to -14) depending on the length of the side chains. Among these, particularly menaquinone-7 (hereinafter, simply referred to as xe2x80x9cMK-7xe2x80x9d) is a representative vitamin K2 substance. MK-7 can be primarily synthesized by Bacillus natto, but its isolation is very difficult, because the vitamin K2 content in natto is as small as several to dozen ppm and the half-life of the vitamin K2 is short. The preparation of lipid with high MK-7 content is described probably only in Japanese Unexamined Patent Application Publication No. 8-73396.
For these reasons, production of vitamin K2 in a large amount has been attempted using microorganisms such as Bacillus natto. For the production of natural vitamin K2, many studies are known. For example, the following methods are mentioned: a method for collecting vitamin K2 from a culture of a microorganism belonging to the genus Flavobacterium (Japanese Examined Patent Application Publication Nos. 7-28748 and 7-51070); a method for producing vitamin K by inoculating Bacillus natto into soybean broth, bean curd refuse or the like supplemented with glycerol to cause fermentation (Japanese Unexamined Patent Application Publication No. 10-295393); and a method for producing vitamin K through fermentation with Bacillus natto under conditions of specific culture temperature and time (Japanese Unexamined Patent Application Publication Nos. 8-9916 and 8-19378). In addition to these methods, a method has been proposed in which a fermentation product is extracted with an organic solvent such as an alcohol, an ether, an ester or a ketone to thereby prepare a concentrated lipid which is rich in natural vitamin K2, particularly natural MK-7 (Japanese Unexamined Patent Application Publication No. 8-73396).
However, in the method using a vitamin K-producing bacterium such as Flavobacterium disclosed in Japanese Examined Patent Application Publication Nos. 7-28748 and 7-51070, there is such a problem that a culture or culture broth of Flavobacterium cannot be used in itself as a food additive or a food product, because the safety of Flavobacterium as a food is not yet demonstrated. In the method employing the culture of Bacillus natto in soybean broth, bean curd refuse or the like supplemented with glycerol as disclosed in Japanese Unexamined Patent Application Publication No. 10-295393, although a culture with relatively high vitamin K content of up to 40 mg/liter of culture medium can be certainly produced, about 3 to 10% by weight of glycerol may be contaminated in the culture medium and, therefore, glycerol would inevitably remain in the culture. Accordingly, there is such a problem that, although the raw materials of the culture medium (e.g., soybean broth, bean curd refuse) and Bacillus natto before cultivation can be used for food, the culture or culture broth given after the cultivation cannot be used in itself as a food additive or for food due to the presence of glycerol therein. In the methods employing the fermentation with Bacillus natto under conditions of specific culture temperature and time as disclosed in Japanese Unexamined Patent Application Publication Nos. 8-9916 and 8-19378, a culture with very high vitamin K content of up to about 53 mg/kg soybean and up to about 117 mg/liter culture can be produced, respectively. However, particularly in the method of Japanese Unexamined Patent Application Publication No. 8-9916, the resulting culture is not suitable for a food material because of the smell of ammonia, as described in its specification. In the method disclosed in Japanese Unexamined Patent Application Publication No. 8-19378, although a culture with a high vitamin K content can be produced, most of the produced vitamin K substances are not water-soluble but fat-soluble and, therefore, the use of the vitamin K substances is markedly limited.
The lipid with a high natural menaquinone-7 content prepared by extraction of the fermentation product of Bacillus natto with an organic solvent, as disclosed in Japanese Unexamined Patent Application Publication No. 8-73396 is disadvantageous. This is because, although the lipid with high natural menaquinone-7 content can be produced from a raw material (e.g., soybean) applicable for food, the organic solvent should be removed from the lipid completely upon use in a food or as a food, which requires additional facility or time for the removal. Moreover, as mentioned above, the resulting lipid containing menaquinone-7 is fat-soluble as apparent from its name and, therefore, may be limited in use.
As mentioned above, there have been reported the methods for producing vitamin K or menaquinone-7 from natto or a fermentation product of a by-product of the production process for natto (e.g., refuse, broth) with a microorganism (e.g., Bacillus natto). However, little report has been found concerning a microorganisms itself which can produce vitamin K or a derivative thereof applicable to food in a high yield.
Accordingly, the object of the present invention is to provide a microorganism capable of producing vitamin K or a derivative thereof with good efficiency.
The object of the present invention can be achieved by using Yunnan strain SL-001 (FERM BP-6713) which belongs to Bacillus subtilis.